The invention relates to methods of inhibiting the activity of membrane-type matrix metalloproteinase (MT-MMP) in biological systems. More specifically, the invention relates to the use of tetracycline compounds for the inhibition of MT-MMP in mammals.
Matrix metalloproteinases MMPs, matrixins) are a family of related proteolytic enzymes expressed by a wide variety of cells and tissue types in both normal and abnormal situations. These zinc-binding metalloendopeptidase enzymes are unified as a family by virtue of structural homologies, and by proteolytic activity against many components of the extracellular matrix (ECM). As a result, the MMPs play important roles in development and morphogenesis as well as in tissue remodeling following injury. In particular, matrix metalloproteinases are distinguished from other metalloproteinases by virtue of their susceptibility to activation of the zymogen by thiol-modifying reagents (e.g., 4-aminophenyl mercuric acetate (APMA) and other mercurial compounds, N-ethylmaleimide, oxidized glutathione), their inhibition by a group of endogenous substances known collectively as "tissue inhibitors of metalloproteinases" or "TIMPs," and the presence of a consensus sequence in the propeptide forms. Nagase (1996).
However, the MMPs have widely divergent substrate specificities and activities, leading to their classification into a series of subtypes. The collagenases include interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13), and are characterized by their ability to specifically degrade triple helical regions of collagens I, II, and II. The gelatinases include gelatinase A (MMP-2; 72-kDa gelatinase) and gelatinase B (MMP-9; 92-kDa gelatinase), and are characterized by their ability to specifically cleave gelatins, but they are further characterized by their ability to hydrolyze collagen IV, elastin and other substrates. The stromelysins include stromelysin 1 (MMP-3), stromelysin 2 (MMP-10), and in certain systems stromelysin 3 (MMP-1). The stromelysins 1 and 2 characteristically degrade a variety of substrates including aggrecan, fibronectin, laminin, numerous collagen types, and play a role in activation of other enzymes.
The MMP family also includes several other MMPs that do not neatly fit into the collagenase, gelatinase, and stromelysin subfamilies. Matrilysin (MMP-7; PUMP-1) differs substantially in structure, lacking a C-terminal domain common to the other MMPs. Metalloelastase (MMP-12) is characterized by its ability to degrade elastin.
A more recently discovered MMP that is substantially distinct from the conventional MMP structural subfamilies is membrane-type matrix metalloproteinase (MT-MMP). MT-MMP differs from other MMPs in a number of ways. For example, most MMPs are secreted by the cells in which they are made, but MT-MMP, as is implicit in its name, is expressed as a membrane-bound protein on the surface of the cell. It is believed that this feature is unique among the MMPs and that such expression may be attributable to a hydrophobic domain of up to 24 amino acids. See Nagase (1996).
The MT-MMP enzyme also has characteristic substrate specificity, with particular capacity for converting pro-MMP-2 and pro-MMP-9 into the active forms of these gelatinases (Murphy et al. 1992; Zucker et al. 1995). However, MT-MMP is also characterized by direct degradation of gelatin. MT-MMP may further act as a surface receptor for TIMP-2, and the resulting MT-MMP/TIMP-2 complex may then act as a receptor for pro-gelatinase A, which can activate the gelatinase (Strongin et al. 1995; Cao et al. 1996; Lichte et al. 1996). This plasma membrane-activated MMP-2 can in turn activate pro-MMP-9 and this process is inhibited by TIMPs (Fridman et al. 1995).
Unlike other MMPs, MT-MMPs and MMP-1 (stromelysin-3) are activated by an intracellular proteinase, furin, a Golgi-associated serine proteinase (Pei and Weiss 1995). Similar to MMP-11, the mechanism of activation of MT-MMP is under investigation and may also involve an intracellular activation through a sequence which can be recognized by furin-like enzymes (Sato et al. 1996). Recently, Cao et al. (1996) demonstrated that the 63 kDa pro-MT-MMP expressed on the cell surface has not been processed (contransfection with furin did not change the molecular weight of MT-MMP). These authors hypothesize that conformational effects induced by the plasma membrane localization of MT-MMP may provide functional activity to pro-MMP-2 without cleavage of the molecule. Receptor-bound TIMP-2 may participate in this process.
Investigators have identified MT-MMP expression by normal tissues, e.g., placenta, lung, and kidney (Takino et al. 1994). A form of MT-MMP, identified as membrane-type 1 MMP (MT.sub.1 -MMP) has also been found to be expressed by osteoclasts, the principal cell type involved in bone resorption (Sato et al 1997). Other investigators have identified three different forms of MT-MMP expressed by rat smooth muscle cells, that appear to be involved in matrix remodeling of blood vessels (Shofuda et al 1997).
Lee et al. (1997) have observed expression of a gelatinase activator that appears to localize to the Golgi membranes. Lee et al. hypothesize that this activator may be MT-MMP, and that a change in localization of this activator to the plasma membrane may occur in tumor cells. Other investigators have identified excessive MT-MMP expression by lung carcinomas (Sato et al. 1994). Still others have seen MT-MMP expression by fibroblasts in tumor stroma of colon, breast, head and neck carcinomas (Okada et al. 1995).
MT-MMP expressed on cancer cell membranes also is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases (i.e., digestion of type I, II, and III collagens into characteristic 3/4 and 1/4 fragments) (Ohuchi et al. 1997; Cao et al. 1998). As noted above, MT-MMP also exhibits gelatinolytic activity, as measured by gelatin zymography (Imai et al. 1996). Thus, MT-MMP may play a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of pro-MMP-2, which is produced constitutively in relatively high concentrations by many cell types including tumor cells (Sato et al. 1994; Sato et al. 1997).
Tetracycline and a number of its chemical relatives form a particularly successful class of antibiotics. Certain of the tetracycline compounds, including tetracycline itself, as well as sporocycline, etc., are broad spectrum antibiotics, having utility against a wide variety of bacteria. The parent compound, tetracycline, has the following general structure: ##STR1##
The numbering system for the multiple ring nucleus is as follows: ##STR2##
Tetracycline, as well as the 5--OH (terramycin) and 7--Cl (aureomycin) derivatives, exist in nature, and are all well known antibiotics. Semisynthetic derivatives such as 7-dimethylamino-tetracycline (minocycline) and 6.alpha.-deoxy-5-hydroxy-tetracycline (doxycycline) are also known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements of the structure must be retained to do so. Recently, however, a class of compounds has been defined that are structurally related to the antibiotic tetracyclines, but which have had their antibiotic activity substantially or completely expunged by chemical modification. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher (1978). According to Mitscher, modification at positions 5-9 of the tetracycline ring system can be made without causing the complete loss of antibiotic properties.
However, changes to the basic structure of the ring system, or replacement of substituents at positions 1-4 or 10-12, generally lead to synthetic tetracyclines with substantially less, or essentially no, antibacterial activity. For example, 4-de(dimethylamino)tetracycline is commonly considered to be a non-antibacterial tetracycline. These compounds, known as chemically-modified tetracyclines (CMTs) have been found to possess a number of interesting properties, such as the inhibition of excessive collagenolytic activity both in vitro and in vivo. See, for example, Golub et al. (1991).
More recently, it has been established that tetracyclines, which are rapidly absorbed and have a prolonged plasma half-life, exert biological effects independent of their antimicrobial activity (Golub et al. 1991, Golub et al. 1992, Uitto et al. 1994). Such effects include inhibition of some but not all matrix metalloproteinases. Specific activity has been shown with respect to collagenases (MMP-1; MMP-8; MMP-13) and gelatinases (MMP-2; MMP-9), as well as prevention of pathologic tissue destruction (Golub et al. 1991). However, Applicants are not aware of any evidence in the prior art disclosing or suggesting that tetracycline compounds could be of any use in inhibiting MT-MMP activity, either in normal systems or in systems characterized by abnormal or excessive MT-MMP activity.
Recent studies have suggested that, in some systems, certain tetracyclines and inhibitors of metalloproteinases can inhibit tumor progression (DeClerck et al. 1994) or angiogenesis (WIPO publication WO 92/12717; Maragoudakis et al. 1994). Zucker et al. (1985) showed that minocycline can inhibit melanoma cell activity in vitro. Some tetracyclines may exhibit cytostatic effects against some tumors (Kroon et al. 1984; van den Bogert et al. 1986). Pro-gelatinase A (MMP-2) has been reported to be associated with tumor spread (Yu et al. 1997). 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) has been shown to experimentally suppress prostate and melanoma tumor growth and metastasis in vivo (Lokeshwar et al. 1998; Seftor et al. 1998).
While tetracycline antibiotics are generally effective for treating infection, the use of these compounds can lead to undesirable side effects. For example, the long term administration of antibiotic tetracyclines can reduce or eliminate healthy microbial flora, such as intestinal flora, and can lead to the production of antibiotic resistant organisms or the overgrowth of yeast and fungi. Accordingly, chemically-modified tetracyclines, in which the antimicrobial activity is attenuated or deleted, can be preferred for use in applications in which anti-collagenolytic activity is indicated.
In view of the above considerations, it is clear that there is a need to supplement existing methods of inhibiting proteinase-mediated physiological and structural changes in biological systems, especially as therapeutic interventions in disease processes. It is also apparent that there is a need for advanced and precise inhibitors of MT-MMP activity in diagnostic and therapeutic applications, especially in respect of those medical conditions that are characterized by excessive MT-MMP activity. In particular, it is desirable to identify new and selective MT-MMP inhibitors with relatively high activity, i.e., being active at doses that are substantially free of harmful side effects.
Accordingly, it is one of the purposes of this invention to overcome the above limitations in modulation or control of proteinase activity, by providing a compound and method for inhibiting membrane-type matrix metalloproteinase, and inhibiting deleterious effects of excessive activity of the enzyme in biological systems. In particular, it is a one of the purposes of the invention to provide new and selective MT-MMP inhibitors that are active at doses that are substantially free of harmful side effects.